Cgrp receptor antagonists

ABSTRACT

The disclosures herein relate to novel compounds of formula 
     
       
         
         
             
             
         
       
     
     wherein Ar 1  and R 1  are as defined herein, and their use in treating, preventing, ameliorating, controlling or reducing cerebrovascular or vascular disorders associated with CGRP receptor function.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of Great Britain Patent Application No. 1519194.3, filed Oct. 30, 2015, the entirety of which is incorporated by reference herein.

TECHNICAL FIELD

This application relates to novel compounds and their use as CGRP receptor antagonists. Compounds described herein may be useful in the treatment or prevention of cerebrovascular or vascular disorders such as migraine. The application is also directed to pharmaceutical compositions comprising these compounds and the manufacture and use of these compounds and compositions in the prevention or treatment of such cerebrovascular or vascular disorders.

BACKGROUND OF THE INVENTION

Migraine is a highly disabling neurovascular disorder characterized by attacks of moderate to severe headache that are often associated with nausea, vomiting, photophobia, and phonophobia. The attacks can last from 4 to 72 h, and the average attack frequency is 1 or 2 per month. About 20-30% of migraine patients experience transient focal neurologic symptoms known as aura, which are usually visual and can precede or accompany the headache. Migraine afflicts about 11% of adults worldwide and results in a significant socioeconomic burden, in terms of both quality of life and lost productivity.

Whilst the pathomechanism of migraine is still unclear, one of the leading hypotheses is based on activation of the trigeminovascular system (TS). Several neuropeptides participate in this activation, calcitonin gene-related peptide (CGRP) playing a crucial role among them. CGRP exerts various biological effects through the peripheral and central nervous system (CNS). The functional CGRP-receptor (CGRP-R) complex has been well characterized, and novel therapeutic approaches target CGRP itself and its receptors. This invention relates to the development of CGRP receptor antagonists (CGRP-RA).

CGRP, a 37-amino acid neuropeptide derived from the gene encoding calcitonin, is formed from the alternative splicing of the calcitonin/CGRP gene located on chromosome 11. In humans, CGRP has two isoforms: α- and β-CGRP. The β-isoform differs from the α-isoform in the amino acids located at positions 3, 22 and 25. The chemical structure of CGRP involves a disulphide bridge between residues 2 and 7 and an amidated C-terminus. The cyclic cysteine2-cysteine7 motif has a basic role in receptor activation. In the human trigeminal ganglia (TRIG), CGRP-immunoreactive neurons account for up to 50% of all neurons. It has been demonstrated through an in situ hybridization technique that 40% of all nerve cell bodies contain CGRP mRNA and CGRP. Double immunostaining has shown that in the human TRIG CGRP is co-localized with nitric oxide synthase, substance P (SP), pituitary adenylate cyclase activating peptide (PACAP) and nociceptin, which may play a role in the pathomechanism of migraine.

The functional CGRP-R consists of three proteins: i) Calcitonin Receptor Like Receptor (known as CRLR, CALCRL or CLR) is a seven-transmembrane spanning protein, which forms the ligand binding site with; ii) RAMP1, determining the specificity of the receptor; and iii) the CGRP-R component protein (RCP) couples the receptor to intracellular signal transduction pathways and to adenylyl cyclase.

It is thought that the C-terminal region of CGRP initially binds to the large N-terminal extracellular domain (ECD) of the receptor, likely making interactions with both CLR and RAMP1. This initial binding event greatly increases the local concentration of the N-terminal region of CGRP in the vicinity of the juxtamembrane portion of CLR, allowing their relatively weak interaction to occur and resulting in receptor activation. Since mutagenesis experiments indicated that most small molecule antagonists interacted with the ECD of CLR/RAMP1, it was hypothesized that they bind to this region of the receptor and prevent the initial binding of CGRP to the receptor. A notable exception to this model of peptide binding and small molecule receptor antagonism is the hydroxypyridine class of antagonists, which apparently interact with transmembrane domain 7 (TM7) in CLR and not with the extracellular domain (Bell I M, J. Med. Chem., 2014, 57(19), 7838-58).

The first clinically tested CGRP-RA, olcegepant, was based on a dipeptide backbone, had high molecular weight, and was not orally bioavailable. Nonetheless, when dosed intravenously, olcegepant proved to be an effective antimigraine agent, and this proof-of-concept study greatly increased interest in the field. Following the success of olcegepant, a number of orally acting CGRP-RAs were advanced to clinical trials. Telcagepant and compounds BI 44370, MK-3207, and BMS-927711 have all been used for acute treatment of migraine as oral agents. Taken together, the results from these clinical studies demonstrate that CGRP-RAs can exhibit similar antimigraine efficacy to the gold standard triptan drugs but with a significantly lower incidence of adverse events than is typically observed with a triptan. It is worth noting that the available data indicate that these CGRP blockers do not cause vasoconstriction and suggest that they may have a superior cardiovascular safety profile to the triptans. One potential concern that has been reported with some CGRP-RAs is the observation of elevated levels of liver transaminases in some patients, and this reportedly led to the discontinuation of MK-3207. Although elevated liver enzymes were also found in a small number of subjects after dosing with telcagepant for an extended period, it is not clear if these findings are in some way mechanism-based or specific to these two compounds. In clinical trials for acute migraine therapy, the CGRP-RAs displayed favorable effects, but their frequent administration was associated with liver toxicity (the elevation of liver transaminases), which limited their clinical use. Hence, there is a need to develop new CGRP-RAs which do not induce liver injury.

SUMMARY OF THE INVENTION

One possibility to address the risk of liver injury is to target a non-oral route of delivery for a small molecule which will place a lower burden on the liver through first-pass exposure. The compounds of the invention can be used for sub-cutaneous, intravenous and/or intranasal routes of administration. The molecular profile for a CGRP-RA intended for such routes of administration differs from the profile required for an oral molecule: extremely high affinity and functional potency, coupled with extremely high solubility is required. Disclosed herein are novel compounds, and the first medical use of said compounds as CGRP receptor antagonists.

Compounds of the invention include compounds of formula (I)

or salts thereof, wherein R¹ is H or F and Ar¹ is an optionally substituted 5 membered heterocyclic ring containing at least two nitrogen atoms.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to novel compounds. The invention also relates to the use of novel compounds as CGRP receptor antagonists. The invention further relates to the use of compounds in the manufacture of medicaments for use as CGRP receptor antagonists. The invention further relates to compounds, compositions and medicaments for the treatment of cerebrovascular or vascular disorders such as migraine (including subtypes such as: migraine without aura, chronic migraine, pure menstrual migraine, menstrually-related migraine, migraine with aura, familial hemiplegic migraine, sporadic hemiplegic migraine, basilar-type migraine, cyclical vomiting, abdominal migraine, benign paroxysmal vertigo of childhood, retinal migraine), status migrainosus, cluster headache, dialysis headache, paroxysmal hemicrania, osteoarthritis, hot flashes associated with menopause or medically induced menopause due to surgery or drug treatment, hemicrania continua, cyclic vomiting syndrome, allergic rhinitis, or rosacea. The invention further relates to compounds, compositions and medicaments for the treatment of broader pain states and diseases involving neurogenic inflammation including dental pain, earache, middle ear inflammation, sunburn, joint pain associated with osteoarthritis and rheumatoid arthritis, cancer pain, fibromyalgia, diabetic neuropathy, pain associated with inflammatory bowel disease—Crohn's disease, gout, complex regional pain syndrome, Behçet's disease, endometriosis pain, back pain or cough.

Compounds exemplified herein are based around the structure of formula (I):

or salts thereof, wherein R¹ is H or F and Ar¹ is an optionally substituted 5 membered heterocyclic ring containing at least two nitrogen atoms.

In a particular embodiment, Ar¹ is an optionally substituted five-membered heterocyclic ring including at least two nitrogen atoms, wherein the optional substituents are selected from (C₁-C₆)alkyl, CO₂R² where R² is H or (C₁-C₃)alkyl.

In a particular embodiment, R¹ is H.

In a more particular embodiment Ar¹ is a five-membered heterocyclic ring including two or three nitrogen atoms, optionally substituted with (C₁-C₆)alkyl.

In a particular embodiment, Ar¹ is selected from:

Further embodiments of the invention include methods of treatment comprising administering a compound of formulas (I) as a CGRP receptor antagonist. The treatment using a compound of formulas (I) may be in the treatment of cerebrovascular disorders such as migraine (including subtypes such as: migraine without aura, chronic migraine, pure menstrual migraine, menstrually-related migraine, migraine with aura, familial hemiplegic migraine, sporadic hemiplegic migraine, basilar-type migraine, cyclical vomiting, abdominal migraine, benign paroxysmal vertigo of childhood, retinal migraine), status migrainosus, cluster headache, dialysis headache, paroxysmal hemicrania, osteoarthritis, hot flashes associated with menopause or medically induced menopause due to surgery or drug treatment, hemicrania continua, cyclic vomiting syndrome, allergic rhinitis, or rosacea. The invention further relates to compounds, compositions and medicaments for the treatment of broader pain states and diseases involving neurogenic inflammation including dental pain, earache, middle ear inflammation, sunburn, joint pain associated with osteoarthritis and rheumatoid arthritis, cancer pain, fibromyalgia, diabetic neuropathy, pain associated with inflammatory bowel disease—Crohn's disease, gout, complex regional pain syndrome, Behçet's disease, endometriosis pain, back pain or cough.

Certain novel compounds of the invention show particularly high activities as CGRP receptor antagonists. Exemplary compounds include:

The NMR and LCMS properties as well as the biological activities of these compounds are set out in Tables 2 and 3.

To the extent that any of the compounds described have chiral centres, the present invention extends to all optical isomers of such compounds, whether in the form of racemates or resolved enantiomers. The invention described herein relates to all crystal forms, solvates and hydrates of any of the disclosed compounds however so prepared. To the extent that any of the compounds and intermediates disclosed herein have acid or basic centres such as carboxylates or amino groups, then all salt forms of said compounds are included herein. In the case of pharmaceutical uses, the salt should be seen as being a pharmaceutically acceptable salt.

Pharmaceutically acceptable salts that may be mentioned include acid addition salts and base addition salts. Such salts may be formed by conventional means, for example by reaction of a free acid or a free base form of a compound with one or more equivalents of an appropriate acid or base, optionally in a solvent, or in a medium in which the salt is insoluble, followed by removal of said solvent, or said medium, using standard techniques (e.g. in vacuo, by freeze-drying or by filtration). Salts may also be prepared by exchanging a counter-ion of a compound in the form of a salt with another counter-ion, for example using a suitable ion exchange resin.

Examples of pharmaceutically acceptable salts include acid addition salts derived from mineral acids and organic acids, and salts derived from metals such as sodium, magnesium, or preferably, potassium and calcium.

Examples of acid addition salts include acid addition salts formed with acetic, 2,2-dichloroacetic, adipic, alginic, aryl sulfonic acids (e.g. benzenesulfonic, naphthalene-2-sulfonic, naphthalene-1,5-disulfonic and p-toluenesulfonic), ascorbic (e.g. L-ascorbic), L-aspartic, benzoic, 4-acetamidobenzoic, butanoic, (+)-camphoric, camphor-sulfonic, (+)-(1S)-camphor-10-sulfonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulfuric, ethane-1,2-disulfonic, ethanesulfonic, 2-hydroxyethanesulfonic, formic, fumaric, galactaric, gentisic, glucoheptonic, gluconic (e.g. D-gluconic), glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), α-oxoglutaric, glycolic, hippuric, hydrobromic, hydrochloric, hydriodic, isethionic, lactic (e.g. (+)-L-lactic and (±)-DL-lactic), lactobionic, maleic, malic (e.g. (−)-L-malic), malonic, (±)-DL-mandelic, metaphosphoric, methanesulfonic, 1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, L-pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulfuric, tannic, tartaric (e.g. (+)-L-tartaric), thiocyanic, undecylenic and valeric acids.

Particular examples of salts are salts derived from mineral acids such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulfuric acids; from organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, arylsulfonic, pamoic acids; and from metals such as sodium, magnesium, or preferably, potassium and calcium.

Also encompassed are any solvates of the compounds and their salts. Preferred solvates are solvates formed by the incorporation into the solid state structure (e.g. crystal structure) of the compounds of the invention of molecules of a non-toxic pharmaceutically acceptable solvent (referred to below as the solvating solvent). Examples of such solvents include water, alcohols (such as ethanol, isopropanol and butanol) and dimethylsulfoxide. Solvates can be prepared by recrystallising the compounds of the invention with a solvent or mixture of solvents containing the solvating solvent. Whether or not a solvate has been formed in any given instance can be determined by subjecting crystals of the compound to analysis using well known and standard techniques such as thermogravimetric analysis (TGE), differential scanning calorimetry (DSC) and X-ray crystallography.

The solvates can be stoichiometric or non-stoichiometric solvates. Particular solvates may be hydrates, and examples of hydrates include hemihydrates, monohydrates and dihydrates.

For a more detailed discussion of solvates and the methods used to make and characterise them, see Bryn et al., Solid-State Chemistry of Drugs, Second Edition, published by SSCI, Inc of West Lafayette, Ind., USA, 1999, ISBN 0-967-06710-3.

“Pharmaceutically functional derivatives” of compounds as defined herein includes ester derivatives and/or derivatives that have, or provide for, the same biological function and/or activity as any relevant compound of the invention. Thus, for the purposes of this invention, the term also includes prodrugs of compounds as defined herein.

The term “prodrug” of a relevant compound includes any compound that, following oral or parenteral administration, is metabolised in vivo to form that compound in an experimentally-detectable amount, and within a predetermined time (e.g. within a dosing interval of between 6 and 24 hours (i.e. once to four times daily)).

Prodrugs of compounds may be prepared by modifying functional groups present on the compound in such a way that the modifications are cleaved, in vivo when such prodrug is administered to a mammalian subject. The modifications typically are achieved by synthesizing the parent compound with a prodrug substituent. Prodrugs include compounds wherein a hydroxyl, amino, sulfhydryl, carboxyl or carbonyl group in a compound is bonded to any group that may be cleaved in vivo to regenerate the free hydroxyl, amino, sulfhydryl, carboxyl or carbonyl group, respectively.

Examples of prodrugs include, but are not limited to, esters and carbamates of hydroxyl functional groups, ester groups of carboxyl functional groups, N-acyl derivatives and N-Mannich bases. General information on prodrugs may be found e.g. in Bundegaard, H. “Design of Prodrugs” p. 1-92, Elsevier, New York-Oxford (1985).

DEFINITIONS

C₁-C₆ Alkyl

Alkyl means an aliphatic hydrocarbon group. The alkyl group may be straight or branched. “Branched” means that at least one carbon branch point is present in the group, for example isopropyl or tertiarybutyl. C₁-C₃ alkyl groups include methyl, ethyl, n-propyl, i-propyl. The alkyl group may be optionally substituted.

Heterocyclic

Heterocyclic means a cyclic group which may be aromatic in which at least one ring member is other than carbon. For example, at least one ring member (for example one, two or three ring members) may be selected from nitrogen, oxygen and sulphur. The point of attachment of heteroaryl groups may be via any atom of the ring system. Exemplary heteroaryl groups include indazolyl, imidazolyl, 1,2,4-triazolyl, quinolin-2(1H)-one, piperidinyl, and the like. Where the heteroaryl group is 5-membered, the heteroaryl group includes imidazolyl, 1,2,4-triazolyl, and the like.

Optionally Substituted

“Optionally substituted” as applied to any group means that the said group may if desired be substituted with one or more substituents, which may be the same or different.

The term “pharmaceutical composition” in the context of this invention means a composition comprising an active agent and comprising additionally one or more pharmaceutically acceptable carriers. The composition may further contain ingredients selected from, for example, diluents, adjuvants, excipients, vehicles, preserving agents, fillers, disintegrating agents, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavouring agents, perfuming agents, antibacterial agents, antifungal agents, lubricating agents and dispersing agents, depending on the nature of the mode of administration and dosage forms. The compositions may take the form, for example, of tablets, dragees, powders, elixirs, syrups, liquid preparations including suspensions, sprays, inhalants, tablets, lozenges, emulsions, solutions, cachets, granules, capsules and suppositories, as well as liquid preparations for injections, including liposome preparations.

The dosages may be varied depending upon the requirements of the patient, the severity of the condition being treated, and the compound being employed. Determination of the proper dosage for a particular situation is within the skill of the art. Generally, treatment is initiated with the smaller dosages which are less than the optimum dose of the compound. Thereafter the dosage is increased by small increments until the optimum effect under the circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day if desired.

The magnitude of an effective dose of a compound will, of course, vary with the nature of the severity of the condition to be treated and with the particular compound and its route of administration. The selection of appropriate dosages is within the ability of one of ordinary skill in this art, without undue burden. In general, the daily dose range may be from about 10 μg to about 30 mg per kg body weight of a human and non-human animal, preferably from about 50 μg to about 30 mg per kg of body weight of a human and non-human animal, for example from about 50 μg to about 10 mg per kg of body weight of a human and non-human animal, for example from about 100 μg to about 30 mg per kg of body weight of a human and non-human animal, for example from about 100 μg to about 10 mg per kg of body weight of a human and non-human animal and most preferably from about 100 μg to about 1 mg per kg of body weight of a human and non-human animal.

Preparation of the Compounds of the Invention

Compounds of the invention may be prepared by routes including those in Scheme 1. Details of many of the standard transformations such as those in the routes below and others which could be used to perform the same transformations can be found in standard reference textbooks such as “Organic Synthesis”, M. B. Smith, McGraw-Hill (1994) or “Advanced Organic Chemistry”, 4^(th) edition, J. March, John Wiley & Sons (1992).

Urea formations between amino acid intermediates, for example methyl esters of amino acids, and amine intermediates can be formed under conditions using a coupling agent such as DSC or CDI in the presence of a base such as triethylamine or DIPEA in solvents such as DMF and/or DCM. The methyl ester portion of the subsequently formed urea derivatives can be saponified using aqueous bases such as lithium hydroxide in a suitable solvent such as THF, MeOH, 1,4-dioxane, or a mixture thereof. The acid intermediates thus formed can be converted into amide examples under standard conditions, for example using a coupling agent such as HATU, in the presence of a base such as DIPEA or triethylamine in a suitable solvent such as DMF. The amine partners for such amide couplings can be prepared using an appropriate combination of standard transformations (for example reductive aminations using an amine, an aldehyde or ketone, and a reducing agent such as sodium triacetoxyborohydride or sodium cyanoborohydride, in a solvent such as MeOH or DCE, optionally in the presence of an additive such as acetic acid or zinc chloride; or alkylation using an alkyl halide and a strong base such as sodium hydride in a suitable solvent such as DMF). Following standard transformations such as the above, or during such a sequence of such transformations, removal of standard protecting groups may be necessary and can be undertaken using conditions which can be found in reference textbooks, for example “Protecting Groups”, 3^(rd) edition, P. J. Kocieński, Georg Thieme Verlag (2005). One such transformation is the removal of a tert-butoxycarbonyl group (commonly known as a Boc group) from an amine under acidic conditions such as HCl in a solvent such as 1,4-dioxane, MeOH, EtOH, DCM or combinations thereof. It can be appreciated that Boc deprotection of amine intermediates of the invention which possess additional basic centres may result in hydrochloride salts of different stoichiometries. For example the Boc deprotection of an intermediate with one additional basic centre will result in the formation of a new amine intermediate which is for example the mono-hydrochloride or di-hydrochloride salt, which will often be used without neutralisation of the hydrochloride salt to produce the free base of the intermediate, as it can be appreciated that in the subsequent amide formation an excess of a base such as DIPEA or triethylamine is typically used to neutralise the hydrochloride salt. Amine intermediates of the invention formed by Boc-deprotection which are used without neutralisation to the free base are named herein as the hydrochloride (x HCl), and the present invention extends to all salt forms of the said intermediates. Examples of the invention may be transformed into further examples using standard transformations such as those detailed above, for example saponification of an ester using conditions such as those detailed above.

General Procedures

Where no preparative routes are included, the relevant intermediate is commercially available. Commercial reagents were utilized without further purification. Room temperature (rt) refers to approximately 20-27° C. ¹H NMR spectra were recorded at 400 MHz or 500 MHz on Bruker, Varian or JEOL instruments. Chemical shift values are expressed in parts per million (ppm), i.e. (δ)-values. The following abbreviations are used for the multiplicity of the NMR signals: s=singlet, br=broad, d=doublet, t=triplet, q=quartet, quin=quintet, h=heptet, dd=doublet of doublets, dt=double of triplets, m=multiplet. Coupling constants are listed as J values, measured in Hz. NMR and mass spectroscopy results were corrected to account for background peaks. Where complex NMR spectra of intermediates or examples exist due to the presence of tautomeric forms data are provided for the major form observed. Chromatography refers to column chromatography performed using silica and executed under positive pressure (flash chromatography) conditions. LCMS experiments were carried out using electrospray conditions under the conditions below. LCMS data are given in the format: Mass ion, electrospray mode (positive or negative), retention time (experimental text and Table 1); Mass ion, electrospray mode (positive or negative), retention time, approximate purity (Table 2).

Method A. Instruments: Hewlett Packard 1100 with G1315A DAD, Micromass ZQ; Column: Waters X-Bridge C-18, 2.5 micron, 2.1×20 mm or Phenomenex Gemini-NX C-18, 3 micron, 2.0×30 mm; Gradient [time (min)/solvent D in C (%)]: 0.00/2, 0.10/2, 8.40/95, 10.00/95; Solvents: solvent C=2.5 L H₂O+2.5 mL 28% ammonia in water solution; solvent D=2.5 L MeCN+135 mL H₂O+2.5 mL 28% ammonia in water solution; Injection volume 1 μL; UV detection 230 to 400 nM; column temperature 45° C.; Flow rate 1.5 mL/min.

Method B. Instruments: Agilent Technologies 1260 Infinity LC with Chemstation software, Diode Array Detector, Agilent 6120B Single Quadrupole MS with API-ES Source; Column: Phenomenex Gemini-NX C-18, 3 micron, 2.0×30 mm; Gradient [time (min)/solvent D in C (%)]: 0.00/5, 2.00/95, 2.50/95, 2.60/5, 3.00/5; Solvents C and D are as described above in Method A; Injection volume 0.5 μL; UV detection 190 to 400 nM; column temperature 40° C.; Flow rate 1.5 mL/min.

Method C. Instruments: Waters Acquity H Class, Photo Diode Array, SQ Detector; Column: BEH C18, 1.7 micron, 2.1×50 mm; Gradient [time (min)/solvent B in A (%)]: 0.00/5, 0.40/5, 0.8/35, 1.20/55, 2.50/100, 3.30/100 4.00/5; Solvents: solvent A=5 mM ammmonium acetate and 0.1% formic acid in H₂O; solvent B=0.1% formic acid in MeCN; Injection volume 2 μL; UV detection 200 to 400 nM; Mass detection 100 to 1200 AMU (+ve electrospray); column at ambient temperature; Flow rate 0.5 mL/min.

Method D. Instruments: Waters Acquity H Class, Photo Diode Array, SQ Detector; Column: X-Bridge C18, 5 micron, 150×4.6 mm; Gradient [time (min)/solvent E in F (%)]: 0.01/10, 5.00/90, 7.00/100, 11.00/100, 11.01/10 12.00/10; Solvents: solvent E=0.1% ammonia in H₂O; solvent F=0.1% ammonia in MeCN; Injection volume 10 μL; UV detection 200 to 400 nM; Mass detection 60 to 1000 AMU (+ve electrospray); column at ambient temperature; Flow rate 1.0 mL/min.

Method E. Instruments: Acquity UPLC coupled with SQD mass spectrometer; Column: Acquity UPLC BEH C18, 1.7 micron, 2.1×50 mm; Gradient [time (min)/solvent B in A (%)]: 0.00/5, 1.50/5, 8.75/80, 9.50/90, 9.80/90, 12.00/5; Solvents: solvent A=10 mM aqueous solution of NH₄HCO₃ (adjusted to pH 10 with ammonia); solvent B=MeCN; Injection volume 2 μL; UV detection 210 to 350 nM; column temperature 40° C.; Flow rate 0.9 mL/min.

ABBREVIATIONS

-   CDI=1,1′-carbonyldiimidazole -   DCE=1,2-dichloroethane -   DCM=dichloromethane -   DIPEA=N,N-diisopropylethylamine -   DMAC=N,N-dimethylacetamide -   DMF=dimethylformamide -   DSC=N,N′-disuccinimidyl carbonate -   DMSO=dimethylsulfoxide -   ES=electrospray -   EtOAc=ethyl acetate -   h=hour(s) -   HATU=1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium     3-oxid hexafluorophosphate -   L=litre -   LC=liquid chromatography -   LCMS=liquid chromatography mass spectrometry -   MeCN=acetonitrile -   min=minute(s) -   MS=mass spectrometry -   NMR=nuclear magnetic resonance -   rcf=relative centrifugal force -   rpm=revolutions per minute -   rt=room temperature -   s=second(s) -   THF=tetrahydrofuran -   TLC=thin-layer chromatography

Prefixes n-, s-, i-, t- and tert- have their usual meanings: normal, secondary, iso, and tertiary.

Synthesis of Intermediates Preparation of Carboxylic Acid Intermediates Typical procedure for the preparation of carboxylic acid intermediates via urea formation and subsequent saponification, as exemplified by the preparation of Intermediate 4, (2R)-3-(7-methyl-1H-indazol-5-yl)-2-({[4-(2-oxo-1,2-dihydroquinolin-3-yl)piperidin-1-yl]carbonyl}amino)propanoic acid

Step 1) To a solution of (R)-methyl 2-amino-3-(7-methyl-1H-indazol-5-yl) propanoate (Intermediate 3, 6.05 g, 25.9 mmol) in DMF (60 mL) under N₂ at approximately −20° C. was added CDI (8.40 g, 51.8 mmol) and the mixture was stirred for 15 min while keeping the temperature below −10° C. A solution of H₂O (2.34 mL) in a few mL of DMF was added and stirring continued for 15 min while keeping the temperature below −10° C. 3-(Piperidin-4-yl)quinolin-2(1H)-one (Intermediate 1, 6.99 g, 30.6 mmol), DIPEA (4.93 mL, 28.2 mmol) and DCM (20 mL) were then added in that order and the mixture was heated to 40° C. under N₂ for 12 h. After cooling to rt, 2M HCl (aq) (38.7 mL) was added and the mixture was extracted twice with DCM. The combined organic extracts were washed three times with H₂O, dried (Na₂SO₄) and concentrated in vacuo. Purification by flash chromatography, eluting with MeOH/DCM (5:95), yielded methyl (2R)-3-(7-methyl-1H-indazol-5-yl)-2-({[4-(2-oxo-1,2-dihydroquinolin-3-yl)piperidin-1-yl]carbonyl}amino)propanoate (10.4 g, 21.3 mmol) as a light tan solid.

¹H NMR: (400 MHz, CDCl₃) δ: 1.40-1.60 (m, 2H), 1.95-1.97 (m, 2H), 2.46 (s, 3H), 2.90-3.00 (m, 2H), 3.11-3.26 (m, 3H), 3.76 (s, 3H), 4.07-4.12 (m, 2H), 4.86-4.91 (m, 1H), 5.18 (d, J=7.6, 1H), 6.93 (s, 1H), 7.17-7.21 (m, 1H), 7.24 (s, 1H), 7.32 (s, 1H), 7.43-7.54 (m, 3H), 7.95 (s, 1H), 10.70 (s, 2H).

Step 2) To a solution of methyl (2R)-3-(7-methyl-1H-indazol-5-yl)-2-({[4-(2-oxo-1,2-dihydroquinolin-3-yl)piperidin-1-yl]carbonyl}amino)propanoate (9.79 g, 20.1 mmol) in 1,4-dioxane (150 mL) was added a solution of LiOH.H₂O (1.26 g, 30.0 mmol) in H₂O (150 mL) and the mixture was stirred at rt for 2 h. The reaction mixture was concentrated in vacuo to near-dryness and re-dissolved in H₂O before being acidified with aqueous 2M HCl (approximately 15 mL) whilst being rapidly stirred. The resulting thick white precipitate was isolated by filtration and washed with H₂O until the washings were near neutral pH. Drying in vacuo yielded the title compound (8.11 g, 17.1 mmol) as an off-white solid.

Data in Table 1. Intermediate 5, (2R)-2-({[4-(7-fluoro-2-oxo-1,2-dihydroquinolin-3-yl)piperidin-1-yl]carbonyl}amino)-3-(7-methyl-1H-indazol-5-yl)propanoic acid

Step 1) Et₃N (1.25 mL, 9.0 mmol) was added to a solution of (R)-methyl 2-amino-3-(7-methyl-1H-indazol-5-yl)propanoate (Intermediate 3, 700 mg, 3.0 mmol) and DSC (845 mg, 3.3 mmol) in DMF (20 mL) and the mixture stirred at rt for 30 min. 7-Fluoro-3-(piperidin-4-yl)quinolin-2(1H)-one hydrochloride (Intermediate 2, 933 mg, 3.3 mmol) was then added portionwise and the reaction mixture stirred at rt overnight before concentration in vacuo. The residue was partitioned between H₂O and DCM, with a small amount of MeOH added to aid dissolution, and the organic phase with washed with H₂O. After concentration in vacuo the residue was purified by flash chromatography, eluting with EtOAc in MeOH (20:1), to yield methyl (2R)-2-({[4-(7-fluoro-2-oxo-1,2-dihydroquinolin-3-yl)piperidin-1-yl]carbonyl}amino)-3-(7-methyl-1H-indazol-5-yl)propanoate (462 mg, 0.91 mmol) as a yellow solid.

LCMS (Method A): m/z 506.3 (ES+), at 3.35 min.

¹H NMR: (400 MHz, DMSO-d₆) δ: ppm 1.22-1.37 (m, 2H), 1.73 (t, J=10.2, 2H), 2.47 (s, 3H), 2.64-2.80 (m, 2H), 2.82-2.94 (m, 1H), 2.95-3.11 (m, 2H), 3.59 (s, 3H), 4.08 (d, J=12.5, 2H), 4.21-4.33 (m, 1H), 6.85 (d, J=7.8, 1H), 6.97-7.10 (m, 3H), 7.41 (s, 1H), 7.59 (s, 1H), 7.70 (dd, J=8.2, 6.2, 1H), 7.87-8.10 (m, 1H), 11.85 (s, 1H), 13.04 (s, 1H)

Step 2) Methyl (2R)-2-({[4-(7-fluoro-2-oxo-1,2-dihydroquinolin-3-yl)piperidin-1-yl]carbonyl}amino)-3-(7-methyl-1H-indazol-5-yl)propanoate (462 mg, 0.91 mmol) was dissolved in THF (6 mL) and MeOH (1.2 mL) and an aqueous solution of LiOH (1M, 1.82 mL, 1.82 mmol) was added dropwise. After stirring at rt for 4 h the reaction mixture was concentrated under a stream of nitrogen, the residue dissolved in a minimum volume of H₂O and acidified with 1M HCl. The resulting precipitate was isolated by filtration, washed with cold H₂O and Et₂O to yield the title compound (406 mg, 0.83 mmol) as a pale yellow solid.

Data in Table 1. Preparation of Amine Intermediates Intermediate 8, 4-(4-methyl-4H-1,2,4-triazol-3-yl)-1,4′-bipiperidine hydrochloride

Step 1) tert-Butyl 4-oxopiperidine-1-carboxylate (Intermediate 7, 2.09 g, 10.5 mmol) was added to a suspension of 4-(4-methyl-4H-1,2,4-triazol-3-yl)piperidine (Intermediate 6, 1.66 g, 10.0 mmol) in DCE (60 mL). The mixture was stirred at rt for 30 min before the addition of sodium triacetoxyborohydride (2.97 g, 14.0 mmol). After stirring at rt overnight tert-butyl 4-oxopiperidine-1-carboxylate (Intermediate 7, 210 mg, 1.05 mmol) was added and the mixture stirred for 8 h at rt, followed by the addition of tert-butyl 4-oxopiperidine-1-carboxylate (Intermediate 7, 600 mg, 3.01 mmol) and sodium triacetoxyborohydride (900 mg, 4.25 mmol) and stirring at rt for 4 d. H₂O (100 mL) was added, the mixture was stirred for 2 min before the phases were separated and the aqueous layer (adjusted to pH 4.7) was washed with DCM (2×30 mL). DCM (30 mL) was added to the aqueous phase, and the aqueous phase was adjusted to pH 7 by the addition of 1M NaOH before extraction with DCM (7×30 mL), adjusting the aqueous phase pH to 7 before each extraction. The aqueous phase was further extracted with DCM (5×30 mL), adjusting the pH to 7.5 before each extraction. The combined organic phases were dried (Na₂SO₄), filtered and concentrated in vacuo to yield tert-butyl 4-(4-methyl-4H-1,2,4-triazol-3-yl)-1,4′-bipiperidine-1′-carboxylate (2.27 g, 6.50 mmol) which was used without purification in the subsequent step.

LCMS (Method E): m/z 350.4 (ES+), at 3.91 min.

¹H NMR: (500 MHz, CD₃OD) δ: ppm 1.43-1.48 (m, 1H), 1.46 (s, 9H), 1.87-2.02 (m, 6H), 2.39-2.46 (m, 2H), 2.51-2.58 (m, 1H), 2.76 (br s, 2H), 2.81-2.92 (m, 1H), 3.10 (dt, J=12.4, 3.6, 2H), 3.71 (s, 3H), 4.14 (dt, J=13.4, 2.4, 2H), 4.59 (br s, 1H), 8.35 (s, 1H)

Step 2) 4N HCl in 1,4-dioxane (16.0 mL, 64.0 mmol) was added to a solution of tert-butyl 4-(4-methyl-4H-1,2,4-triazol-3-yl)-1,4′-bipiperidine-1′-carboxylate (2.27 g, 6.50 mmol) in DCM (120 mL) and the mixture stirred at rt for 2 h. Concentration in vacuo yielded the title compound (2.23 g) as the hydrochloride salt which was used without further purification.

Data in Table 1. Alternative synthesis of Intermediate 8, 4-(4-methyl-4H-1,2,4-triazol-3-yl)-1,4′-bipiperidine

Step 1) Benzyl 4-oxopiperidine-1-carboxylate (Intermediate 22, 23.6 g, 101.1 mmol) and sodium triacetoxyborohydride (28.6 g, 134.8 mmol) were added to a solution of 4-(4-methyl-4H-1,2,4-triazol-3-yl)piperidine (Intermediate 6, 16.0 g, 96.3 mmol) in DCE (580 mL). After stirring at rt overnight acetic acid (5 mL) was added, and after stirring at rt for a further 2 h benzyl 4-oxopiperidine-1-carboxylate (Intermediate 22, 2.35 g, 10.1 mmol) and sodium triacetoxyborohydride (2.86 g, 13.5 mmol) were added. After stirring at rt for 2 h benzyl 4-oxopiperidine-1-carboxylate (Intermediate 22, 7.05 g, 30.3 mmol) and sodium triacetoxyborohydride (8.58 g, 40.4 mmol) were added, the mixture was stirred at rt for 2 h before the addition of benzyl 4-oxopiperidine-1-carboxylate (Intermediate 22, 7.05 g, 30.3 mmol) and sodium triacetoxyborohydride (8.58 g, 40.4 mmol). After stirring at rt overnight H₂O (1 L) was added, the phases were separated, and the aqueous phase was extracted with DCM (3×300 mL). The pH of the aqueous phase was adjusted to 7.5 with 6N aqueous NaOH and extracted with DCM (5×300 mL). The combined organic phases were dried (Na₂SO₄), filtered and concentrated in vacuo to yield benzyl 4-(4-methyl-4H-1,2,4-triazol-3-yl)-1,4′-bipiperidine-1′-carboxylate (23.2 g, 60.6 mmol).

LCMS (Method A): m/z 384.3 (ES+), at 3.29 min.

¹H NMR: (400 MHz, CD₃OD) δ: ppm 1.40-1.52 (m, 2H), 1.86-2.05 (m, 6H), 2.39-2.51 (m, 2H), 2.53-2.66 (m, 1H), 2.75-2.98 (m, 3H), 3.04-3.18 (m, 2H), 3.71 (s, 3H), 4.22 (d, J=13.4, 2H), 5.11 (s, 2H), 7.29-7.39 (m, 5H), 8.35 (s, 1H)

Step 2) A mixture of 10% palladium on carbon (4.76 g) and benzyl 4-(4-methyl-4H-1,2,4-triazol-3-yl)-1,4′-bipiperidine-1′-carboxylate (17.2 g, 44.8 mmol) in EtOH (200 mL) was stirred under an atmosphere of H₂ (1.5 bar) at rt for 90 min. The mixture was filtered through celite, concentrated in vacuo, re-dissolved in DCM (200 mL) and concentrated in vacuo. The re-dissolution and concentration process was repeated five times, and after further drying in vacuo the title compound (10.0 g, 40.1 mmol) was obtained as a white solid.

¹H NMR: (400 MHz, DMSO-d₆) δ: ppm 1.40-1.51 (m, 2H), 1.63-1.77 (m, 4H), 1.80-1.87 (m, 3H), 2.21-2.32 (m, 2H), 2.35-2.45 (m, 1H), 2.52-2.63 (m, 2H), 2.66-2.80 (m, 1H), 2.87-2.99 (m, 2H), 3.09 (d, J=12.5, 2H), 3.59 (br s, 3H), 8.31 (s, 1H).

Intermediate 10, 4-(1H-imidazol-2-yl)-1,4′-bipiperidine hydrochloride

Step 1) A solution of 4-(1H-imidazol-2-yl)piperidine hydrochloride (Intermediate 9, 2.30 g, 12.3 mmol), Et₃N (4.96 mL, 35.6 mmol), tert-butyl 4-oxopiperidine-1-carboxylate (Intermediate 7, 2.44 g, 12.2 mmol) and ZnCl₂ (84.6 mg, 0.61 mmol) in MeOH (100 mL) was stirred at 60° C. for 5 h. After cooling to rt, sodium cyanoborohydride (3.09 g, 49.2 mmol) was added portionwise and the mixture stirred at rt for 16 h. After partitioning between MeOH/DCM (1:9, 300 mL) and H₂O (250 mL) the aqueous phase was extracted with MeOH/DCM (1:9, 300 mL) and the combined organic phases were dried (Na₂SO₄) and concentrated in vacuo to yield tert-butyl 4-(1H-imidazol-2-yl)-1,4′-bipiperidine-1′-carboxylate (5.0 g, colourless oil) which was used without purification in the subsequent step.

TLC: Rf 0.5 (MeOH/DCM 1:9).

LCMS (Method D): m/z 335.2 (ES+), at 4.95 min.

Step 2) Et₃N (4.16 mL, 29.8 mmol) was added to a solution of tert-butyl 4-(1H-imidazol-2-yl)-1,4′-bipiperidine-1′-carboxylate (5.0 g) in DCM (100 mL). After stirring for 15 min di-tert-butyl dicarbonate (4.85 g, 22.2 mmol) was added at 0° C. portion wise and the reaction mixture stirred at rt for 16 h. After partitioning between MeOH/DCM (1:9, 300 mL) and H₂O (250 mL) the aqueous phase was extracted with MeOH/DCM (1:9, 300 mL) and the combined organic phases were dried (Na₂SO₄) and concentrated in vacuo. Purification by gradient flash chromatography, eluting with 0-3% MeOH in DCM yielded tert-butyl 4-[1-(tert-butoxycarbonyl)-1H-imidazol-2-yl]-1,4′-bipiperidine-1′-carboxylate (1.40 g, 3.22 mmol) as a colourless oil.

¹H NMR: (400 MHz, DMSO-d₆) δ: ppm 1.22-1.32 (m, 3H), 1.39 (s, 9H), 1.57 (s, 9H), 1.69-1.72 (m, 4H), 1.85-1.91 (m, 2H), 2.20 (br s, 2H), 2.67 (br d, 3H), 2.99 (br s, 2H), 3.96 (dd, J=10.0, 2H), 6.85 (d, J=1.6, 1H), 7.42 (d, J=1.2, 1H).

Step 3) 4M HCl in 1,4-dioxane (15 mL, 60 mmol) was added dropwise to a solution of tert-butyl 4-[1-(tert-butoxycarbonyl)-1H-imidazol-2-yl]-1,4′-bipiperidine-1′-carboxylate (1.40 g, 3.22 mmol) in 1,4-dioxane (20 mL) at 0° C., and the mixture subsequently stirred at rt for 4 h. After concentration in vacuo trituration with Et₂O yielded the title compound (1.0 g) as an off-white solid.

Data in Table 1. Intermediate 12, 4-(4-methyl-1H-imidazol-5-yl)-1,4′-bipiperidine hydrochloride

Step 1) A mixture of 4-(4-methyl-1H-imidazol-5-yl)piperidine hydrochloride (Intermediate 11, 2.0 g, 9.9 mmol) and triethylamine (1.7 mL, 11.9 mmol) in DMF (40 mL) was stirred at rt. After 5 min tert-butyl 4-oxopiperidine-1-carboxylate (Intermediate 7, 2.4 g, 11.9 mmol) and acetic acid (0.68 ml, 11.9 mmol) were added. After 30 min stirring at rt sodium triacetoxyborohydride (2.52 g, 11.9 mmol) was added. After stirring at rt overnight the reaction mixture was concentrated in vacuo and the residue partitioned between DCM (100 mL) and saturated aqueous NaHCO₃ (100 ml). The aqueous phase was extracted with DCM (100 mL) and the combined organic phases were washed with brine and further dried by passing through a hydrophobic frit. The solvent was removed in vacuo and the product oil crystallised on standing for 3 d. After trituration with diethylether/isohexane the solid was collected by filtration and dried in vacuo to yield tert-butyl 4-(4-methyl-1H-imidazol-5-yl)-1,4′-bipiperidine-1′-carboxylate (1.64 g, 4.7 mmol).

LCMS (Method B): m/z 349.2 (ES+), at 1.24 min.

¹H NMR: (400 MHz, CDCl3) δ: ppm 1.39-1.51 (m, 11H), 1.68-1.91 (m, 6H), 2.20 (s, 3H), 2.24-2.37 (m, 2H), 2.40-2.53 (m, 1H), 2.55-2.77 (m, 3H), 2.97-3.07 (m, 2H), 4.15 (br s, 2H), 7.45 (s, 1H) (1 exchangeable proton not observed)

Step 2) 4N HCl in 1,4-dioxane (10.0 mL, 40.0 mmol) was added to a solution of tert-butyl 4-(4-methyl-1H-imidazol-5-yl)-1,4′-bipiperidine-1′-carboxylate (1.6 g, 4.6 mmol) in DCM (20 mL) and the mixture stirred at rt. After 2 h the reaction mixture was concentrated in vacuo, and the residue taken up and re-evaporated from DCM (×2) to yield the title compound (3.22 g, contains residual solvent) which was used without further purification in the synthesis of Example 2.

Data in Table 1.

A sample of Intermediate 12 (100 mg) was dissolved in DCM (5 mL) and a minimum amount of MeOH, solid NaCO₃ (200 mg) was added and the mixture stirred for 2 h. The reaction mixture was filtered and the filtrate concentrated under a flow of N₂ then in vacuo to yield the title compound as the free base (31 mg, 0.12 mmol).

LCMS (Method A): m/z 249.3 (ES+), at 2.24 min.

¹H NMR: (400 MHz, CD₃OD) δ: ppm 1.47 (qd, J=12.3, 4.1, 2H), 1.72-1.83 (m, 4H), 1.86-1.93 (m, 2H), 2.16 (s, 3H), 2.30-2.39 (m, 2H), 2.46 (tt, J=11.6, 3.5, 1H), 2.53-2.67 (m, 3H), 3.02-3.14 (m, 4H), 7.40 (s, 1H) (2 exchangeable protons not observed).

Intermediate 14, 4-(1-propyl-1H-imidazol-2-yl)piperidine hydrochloride

Step 1) Sodium hydride (60% in mineral oil, 478 mg, 12.0 mmol) was added to a solution of tert-butyl 4-(1H-imidazol-2-yl)piperidine-1-carboxylate (Intermediate 13, 2.50 g, 9.95 mmol) in DMF (50 mL) at 0° C. After stirring at 0° C. for 20 min 1-iodopropane (1.16 mL, 11.9 mmol) was added and the reaction was stirred at rt for 2 h before partitioning between EtOAc (200 mL) and H₂O (150 mL). The aqueous phase was extracted with EtOAc (200 mL) and the combined organic phases were dried (Na₂SO₄), filtered, and concentrated in vacuo. Purification by gradient flash chromatography, eluting with 0-10% MeOH in DCM yielded tert-butyl 4-(1-propyl-1H-imidazol-2-yl)piperidine-1-carboxylate (2.90 g, 9.89 mmol) as a yellow oil.

LCMS (Method C): m/z 294.5 (ES⁺), at 1.77 min.

¹H NMR: (400 MHz, DMSO-d₆) δ: ppm 0.81-0.92 (m, 3H), 1.41 (s, 9H), 1.51-1.70 (m, 6H), 2.89-2.97 (m, 3H), 3.81-3.88 (m, 2H), 3.97-4.00 (d, J=13.6, 2H), 6.78 (s, 1H), 7.04 (s, 1H).

Step 2) The title compound (2.60 g) was prepared from tert-butyl 4-(1-propyl-1H-imidazol-2-yl)piperidine-1-carboxylate (2.90 g, 9.89 mmol) and 4M HCl in 1,4-dioxane (15 mL, 60.0 mmol) using the methods of Intermediate 10, Step 3.

Data in Table 1. Intermediate 15, 4-(1-propyl-1H-imidazol-2-yl)-1,4′-bipiperidine hydrochloride

The title compound (0.70 g, 1.81 mmol) was prepared from 4-(1-propyl-1H-imidazol-2-yl)piperidine hydrochloride (Intermediate 14, 1.00 g) and tert-butyl 4-oxopiperidine-1-carboxylate (Intermediate 7, 868 mg, 4.36 mmol) using the methods of Intermediate 10, Steps 1 and 3.

Data in Table 1. Intermediate 17, ethyl 5-(1,4′-bipiperidin-4-yl)-4H-1,2,4-triazole-3-carboxylate hydrochloride

The title compound was prepared over two steps from ethyl 5-(piperidin-4-yl)-4H-1,2,4-triazole-3-carboxylate (Intermediate 16, 448 mg, 2.00 mmol), tert-butyl 4-oxopiperidine-1-carboxylate (Intermediate 7, 478 mg, 2.40 mmol), sodium triacetoxyborohydride (610 mg, 2.88 mmol) and acetic acid (137 μL, 2.39 mmol) using the methods of Intermediate 12.

Data in Table 1. Intermediate 19, 4-(4H-1,2,4-triazol-3-yl)-1,4′-bipiperidine hydrochloride

Step 1) A mixture of 4-(1H-1,2,4-triazol-5-yl)piperidine hydrochloride (Intermediate 18, 377 mg, 2.00 mmol), tert-butyl 4-oxopiperidine-1-carboxylate (Intermediate 7, 478 mg, 2.40 mmol), acetic acid (137 μL, 2.39 mmol) and Et₃N (279 μL, 2.00 mmol) was stirred at rt for 30 min before addition of sodium triacetoxyborohydride (610 mg, 2.88 mmol) and stirring at rt for 3 d. Further tert-butyl 4-oxopiperidine-1-carboxylate (Intermediate 7, 300 mg, 1.51 mmol) and acetic acid (100 μL, 1.75 mmol) were added and the reaction mixture was stirred at rt for 30 min before addition of sodium triacetoxyborohydride (400 mg, 1.89 mmol) and stirring at rt overnight. After concentration in vacuo purification by gradient flash chromatography, eluting with 0-10% MeOH in DCM, followed by 10% (7N NH₃ in MeOH) in DCM yielded tert-butyl 4-(4H-1,2,4-triazol-3-yl)-1,4′-bipiperidine-1′-carboxylate (350 mg, 1.04 mmol) as a white solid.

LCMS (Method B): m/z 336.2 (ES⁺), at 0.94 min.

¹H NMR: (400 MHz, CD₃OD) δ: ppm, 1.45 (s, 9H), 1.86-1.95 (m, 4H), 2.10-2.13 (m, 2H), 2.56-2.61 (m, 2H), 2.69-2.81 (m, 3H), 2.88-2.94 (m, 1H), 3.03-3.19 (m, 4H), 4.15-4.18 (m, 2H), 8.16 (s, 1H) (one exchangeable proton not observed).

Step 2) The title compound (320 mg) was prepared from tert-butyl 4-(4H-1,2,4-triazol-3-yl)-1,4′-bipiperidine-1′-carboxylate (350 mg, 1.04 mmol) and 4M HCl in 1,4-dioxane (10 mL, 40.0 mmol) in MeOH (10 mL) using the methods of Intermediate 12.

Data in Table 1. Intermediate 21, 4-(5-methyl-4H-1,2,4-triazol-3-yl)-1,4′-bipiperidine hydrochloride

Step 1) A mixture of 4-(3-methyl-1H-1,2,4-triazol-5-yl)piperidine dihydrochloride (Intermediate 20, 478 mg, 2.00 mmol), tert-butyl 4-oxopiperidine-1-carboxylate (Intermediate 7, 478 mg, 2.40 mmol), acetic acid (137 μL, 2.39 mmol) and Et₃N (558 μL, 4.00 mmol) was stirred at rt for 30 min before addition of sodium triacetoxyborohydride (610 mg, 2.88 mmol) and stirring at rt overnight. After concentration in vacuo purification by gradient flash chromatography, eluting with 0-10% (7N NH₃ in MeOH) in DCM yielded tert-butyl 4-(5-methyl-4H-1,2,4-triazol-3-yl)-1,4′-bipiperidine-1′-carboxylate (310 mg, 0.89 mmol) as a colourless solid.

LCMS (Method B): m/z 350.2 (ES⁺), at 1.01 min.

¹H NMR: (400 MHz, CD₃OD) δ: ppm 1.45 (s, 9H), 1.48-1.55 (m, 2H), 1.91-1.99 (m, 7H), 2.11-2.15 (m, 2H), 2.68-2.90 (m, 6H), 3.24-3.27 (m, 2H), 4.16-4.20 (m, 2H) (one exchangeable proton not observed).

Step 2) The title compound (240 mg, 0.93 mmol) was prepared from tert-butyl 4-(5-methyl-4H-1,2,4-triazol-3-yl)-1,4′-bipiperidine-1′-carboxylate (310 mg, 0.89 mmol) and 4M HCl in 1,4-dioxane (5 mL, 20.0 mmol) in MeOH (5 mL) using the methods of Intermediate 12.

Data in Table 1.

Intermediate Name Data 15 4-(1-propyl-1H-imidazol-2-yl)-1,4′- LCMS (Method A): m/z 277.3 (ES+), at 2.81 bipiperidine hydrochloride min. ¹H NMR (400 MHz, CD₃OD) δ: ppm 1.04 (t, J = 7.4, 3H), 1.88-2.00 (m, 2H), 2.08-2.20 (m, 2H), 2.28-2.44 (m, 4H), 2.52 (br d, J = 15.8, 2H), 3.06-3.26 (m, 3H), 3.40-3.50 (m, 2H), 3.57- 3.84 (m, 5H), 4.26 (dd, J = 7.8, 7.0, 2H), 7.59 (d, J = 2.3, 1H), 7.63 (d, J = 2.3, 1H) (exchangeable protons not observed) 16 ethyl 5-(piperidin-4-yl)-4H-1,2,4- Commercially available, triazole-3-carboxylate CAS No. 1513304-93-2 (free base), 1795504- 00-5 (hydrochloride salt) 17 ethyl 5-(1,4′-bipiperidin-4-yl)-4H- LCMS (Method B): m/z 308.2 (ES+), at 0.20 1,2,4-triazole-3-carboxylate min. ¹H NMR (400 MHz, CD₃OD) δ: ppm 1.41 hydrochloride (t, J = 7.1, 3H), 1.99-2.25 (m, 2H), 2.39-2.46 (m, 4H), 3.07-3.18, (m, 3H), 3.29-3.31 (m, 1H) 3.32-3.38 (m, 2H), 3.58-3.68 (m, 4H), 3.71-3.76 (m, 2H), 4.45 (q, J = 7.1, 2H) (exchangeable protons not observed) 18 4-(1H-1,2,4-triazol-5-yl)piperidine Commercially available, CAS No. 893424-25-4 (free base), 1417359-91-1 (hydrochloride salt) 19 4-(4H-1,2,4-triazol-3-yl)-1,4′- LCMS (Method B): m/z 236.2 (ES+), at 0.50 bipiperidine hydrochloride min. ¹H NMR (400 MHz, CD₃OD) δ: ppm 2.08-2.17 (m, 2H), 2.22-2.32 (m, 2H), 2.44-2.54 (m, 4H), 3.11-3.18 (m, 3H), 3.36-3.47 (m, 2H), 3.60-3.67 (m, 3H), 3.75-3.82 (m, 2H), 9.35 (s, 1H) (exchangeable protons not observed) 20 4-(3-methyl-1H-1,2,4-triazol-5-yl) Commercially available, piperidine CAS No. 933713-90-7 (free base), 1221724-59-9 (dihydrochloride salt) 21 4-(5-methyl-4H-1,2,4-triazol-3-yl)- LCMS (Method B): m/z 250.2 (ES+), at 1,4′-bipiperidine hydrochloride 0.68 min. ¹H NMR (400 MHz, CD₃OD) δ: ppm 2.02-2.14 (m, 2H), 2.21-2.31 (m, 2H), 2.42-2.49 (m, 4H), 2.68 (s, 3H), 3.09-3.22 (m, 3H), 3.31- 3.40 (m, 2H), 3.58-3.67 (m, 3H), 3.70-3.78 (m, 2H) (exchangeable protons not observed) 22 benzyl 4-oxopiperidine-1- Commercially available, CAS No. 19099-93-5 carboxylate

Synthesis of Examples

Typical procedures for the preparation of examples via amide coupling, as exemplified by the preparation of the below examples.

Procedure 1: Example 2, N-[(2R)-1-[4-(4-methyl-1H-imidazol-5-yl)-1,4′-bipiperidin-1′-yl]-3-(7-methyl-1H-indazol-5-yl)-1-oxopropan-2-yl]-4-(2-oxo-1,2-dihydroquinolin-3-yl)piperidine-1-carboxamide

A mixture of 4-(4-methyl-1H-imidazol-5-yl)-1,4′-bipiperidine hydrochloride (Intermediate 12, contaminated with solvent residues, assumed to be 21.0 mmol), (2R)-3-(7-methyl-1H-indazol-5-yl)-2-({[4-(2-oxo-1,2-dihydroquinolin-3-yl)piperidin-1-yl]carbonyl}amino)propanoic acid (Intermediate 4, 9.93 g, 21.0 mmol), HATU (8.00 g, 20.9 mmol) and DIPEA (14.6 mL, 83.8 mmol) in DMF (150 mL) was stirred at room temperature overnight before concentration in vacuo. Purification by gradient flash chromatography, eluting with 0-100% solvent B in DCM (where solvent B is 7N NH₃ in MeOH/DCM, 1:9) yielded the title compound (7.50 g, 10.7 mmol) as a white solid.

Data in Table 2. Example 5, 4-(7-fluoro-2-oxo-1,2-dihydroquinolin-3-yl)-N-{(2R)-3-(7-methyl-1H-indazol-5-yl)-1-[4-(4-methyl-4H-1,2,4-triazol-3-yl)-1,4′-bipiperidin-1′-yl]-1-oxopropan-2-yl}piperidine-1-carboxamide

The title compound (45 mg, 0.06 mmol) was prepared from (2R)-2-({[4-(7-fluoro-2-oxo-1,2-dihydroquinolin-3-yl)piperidin-1-yl]carbonyl}amino)-3-(7-methyl-1H-indazol-5-yl)propanoic acid (Intermediate 5, 74 mg, 0.15 mmol), 4-(4-methyl-4H-1,2,4-triazol-3-yl)-1,4′-bipiperidine hydrochloride (Intermediate 8, 97 mg), HATU (69 mg, 0.18) and Et₃N (0.21 mL, 1.51 mmol) in DMF (1.5 mL) using methods of Example 4. The title compound was purified by gradient flash column chromatography eluting with 0-100% solvent B in DCM (where solvent B is 7N NH₃ in MeOH/DCM, 1:9), followed by preparative reversed phase HPLC (Phenomenex Gemini-NX 5 μm C18 column, 100×30 mm, eluting with 10 to 40% MeCN/Solvent B over 12.5 min at 30 mL/min [where solvent B is 0.2% of (28% NH₃/H₂O) in H₂O] and collecting fractions by monitoring at 205 nm).

Data in Table 2. Procedure 2: Example 9, 5-{1′-[(2R)-3-(7-methyl-1H-indazol-5-yl)-2-({[4-(2-oxo-1,2-dihydroquinolin-3-yl)piperidin-1-yl]carbonyl}amino)propanoyl]-1,4′-bipiperidin-4-yl}-4H-1,2,4-triazole-3-carboxylic acid

Lithium hydroxide monohydrate (9 mg, 0.21 mmol) was added to a solution of ethyl 5-{1′-[(2R)-3-(7-methyl-1H-indazol-5-yl)-2-({[4-(2-oxo-1,2-dihydroquinolin-3-yl)piperidin-1-yl]carbonyl}amino)propanoyl]-1,4′-bipiperidin-4-yl}-4H-1,2,4-triazole-3-carboxylate (Example 6, 110 mg, 0.14 mmol) in MeOH (10 mL) and H₂O (2 mL) and the reaction mixture was stirred at rt overnight. Further lithium hydroxide monohydrate (10 mg, 0.24 mmol) was added and after stirring at rt for 1 d the mixture was partially concentrated in vacuo to remove the MeOH. 1N aqueous HCl was added to the residue, which was then concentrated in vacuo and purified by preparative reversed phase HPLC (Phenomenex Gemini-NX 5 μm C18 column, 100×30 mm, eluting with 5 to 35% MeCN/Solvent B over 12.5 min at 30 mL/min [where solvent B is 0.2% of (28% NH₃/H₂O) in H₂O] and collecting fractions by monitoring at 205 nm) to yield the title compound (10 mg, 0.01 mmol) as a colourless solid.

Data in Table 2.

Further examples prepared by the above procedures are detailed in Table 2.

TABLE 2 Ex Intermediates/ LCMS data No. Name Procedure ¹H NMR (Method A) 1 N-[(2R)-1-[4-(1H- 4, 10 (400 MHz, DMSO-d₆) δ: ppm −0.07-0.09 m/z 688.6 (ES⁻), imidazol-2-yl)-1,4′- Procedure 1 (m, 1H), 0.61 (d, J = 8.6, 1H), 1.08-1.92 690.4 (ES⁺) bipiperidin-1′-yl]-3-(7- (m, 10H), 2.00-2.35 (m, 5H), 2.68-2.81 at 2.70 min, 100% methyl-1H-indazol-5-yl)- (m, 3H), 2.81-2.95 (m, 3H), 2.96-3.07 1-oxopropan-2-yl]-4-(2- (m, 1H), 3.30 (s, 3H), 3.83-4.02 (m, 1H), oxo-1,2-dihydroquinolin- 4.05-4.23 (m, 2H), 4.35 (d, J = 11.7, 1H), 3-yl)piperidine-1- 4.76 (q, J = 7.4, 1H), 4.84 (q, J = 7.8, 1H), carboxamide 6.70 (d, J = 7.0, 1H), 6.83 (br s, 2H), 7.01 (s, 1H), 7.07-7.19 (m, 1H), 7.20-7.31 (m, 1H), 7.35 (s, 1H), 7.37-7.48 (m, 2H), 7.56-7.72 (m, 2H), 7.96 (s, 1H), 11.77 (s, 1H), 13.01 (s, 1H) 2 N-[(2R)-1-[4-(4-methyl- 4, 12 (400 MHz, CD₃OD) δ: ppm −0.30 to −0.27 m/z 704.7 (ES⁺) 1H-imidazol-5-yl)-1,4′- Procedure 1 (m, 1H), 0.70-0.77 (m, 1H), 1.24- at 3.69 min, 100% bipiperidin-1′-yl]-3-(7- 1.32 (m, 1H), 1.42-1.94 (m, 11H), 2.17 methyl-1H-indazol-5-yl)- (s, 3H), 2.30-2.57 (m, 7H), 2.84-3.12 (m, 1-oxopropan-2-yl]-4-(2- 7H), 3.94-4.10 (m, 1H), 4.18-4.25 (m, oxo-1,2-dihydroquinolin- 2H), 4.52 (d, J = 13.0, 1H), 5.02-5.08 (m, 3-yl)piperidine-1- 1H), 7.16 (s, 1H), 7.22-7.26 (m, 1H), carboxamide 7.30-7.32 (m, 1H), 7.41 (s, 1H), 7.44- 7.51 (m, 2H), 7.63-7.67 (m, 1H), 7.75 (s, 1H), 8.01 (s, 1H) (4 exchangeable protons not observed) 3 N-{(2R)-3-(7-methyl-1H- 4, 15 (400 MHz, CD₃OD) δ: ppm −0.35 to −0.30 m/z 732.6 (ES⁺) indazol-5-yl)-1-oxo-1-[4- Procedure 1 (m, 1H), 0.77-0.83 (m, 1H), 0.96 (t, at 3.41 min, 95% (1-propyl-1H-imidazol- J = 7.4, 3H), 1.23-2.12 (m, 14H), 2.31- 2-yl)-1,4′-bipiperidin-1′- 2.42 (m, 4H), 2.54-2.58 (m, 3H), 2.86- yl]propan-2-yl}-4-(2- 3.13 (m, 7H), 3.91-3.94 (m, 3H), 4.21- oxo-1,2-dihydroquinolin- 4.25 (m, 2H), 4.49-4.52 (m, 1H), 5.04- 3-yl)piperidine-1- 5.05 (m, 1H), 6.85 (s, 1H), 6.99 (s, 1H), carboxamide 7.15 (s, 1H), 7.22-7.25 (m, 1H), 7.31- 7.33 (m, 1H), 7.44-7.51 (m, 2H), 7.65- 7.66 (m, 1H), 7.75 (s, 1H), 8.00 (s, 1H) (3 exchangeable protons not observed) 4 N-{(2R)-3-(7-methyl-1H- 4, 8 (400 MHz, CD₃OD) δ: ppm −0.30 to −0.25 m/z 705.8 (ES⁺) indazol-5-yl)-1-[4-(4- Procedure 1 (m, 1H), 0.82-0.86 (m, 1H), 1.23- at 3.34 min, 100% methyl-4H-1,2,4-triazol- 2.04 (m, 13H), 2.33-2.45 (m, 3H), 2.57 3-yl)-1,4′-bipiperidin-1′- (s, 3H), 2.65-3.11 (m, 7H), 3.73 (s, 3H), yl]-1-oxopropan-2-yl}-4- 3.94-3.97 (m, 1H), 4.16-4.24 (m, 2H), (2-oxo-1,2- 4.50-4.53 (m, 1H), 5.02-5.08 (m, 1H), dihydroquinolin-3- 7.16 (s, 1H), 7.21-7.25 (m, 1H), 7.32 (d, yl)piperidine-1- J = 7.8, 1H), 7.45-7.50 (m, 2H), 7.63-7.65 carboxamide (m, 1H), 7.73 (s, 1H), 8.00 (s, 1H), 8.36 (s, 1H) (3 exchangeable protons not observed) 5 4-(7-fluoro-2-oxo-1,2- 5, 8 (400 MHz, CD₃OD) δ: ppm −0.45 to −0.23 m/z 723.6 (ES⁺), dihydroquinolin-3-yl)-N- Procedure 1 (m, 1H), 0.61-0.79 (m, 1H), 1.12- at 3.07 min, 100% {(2R)-3-(7-methyl-1H- 1.64 (m, 4H), 1.64-2.00 (m, 8H), 2.07 (d, indazol-5-yl)-1-[4-(4- J = 11.3, 1H), 2.27-2.62 (m, 6H), 2.67 (d, methyl-4H-1,2,4-triazol- J = 9.4, 1H), 2.76-3.15 (m, 6H), 3.61-3.79 3-yl)-1,4′-bipiperidin-1′- (m, 3H), 3.96 (d, J = 13.7, 1H), 4.22 (d, yl]-1-oxopropan-2- J = 13.3, 2H), 4.51 (d, J = 12.9, 1H), 5.00- yl}piperidine-1- 5.14 (m, 1H), 6.94-7.09 (m, 2H), 7.16 (s, carboxamide 1H), 7.38-7.50 (m, 1H), 7.57-7.78 (m, 2H), 7.94-8.06 (m, 1H), 8.30-8.42 (m, 1H) (3 exchangeable protons not observed). 6 ethyl 5-{1′-[(2R)-3-(7- 4, 17 (400 MHz, CD₃OD) δ: ppm −0.33 to −0.29 m/z 763.7 (ES⁺) methyl-1H-indazol-5-yl)- Procedure 1 (m, 1H), 0.83-0.87 (m, 1H), 1.20- at 3.26 min, 95% 2-({[4-(2-oxo-1,2- 2.46 (m, 16H), 2.57 (s, 3H), 2.66-3.11 dihydroquinolin-3- (m, 7H), 3.92-3.99 (m, 1H), 4.21-4.24 yl)piperidin-1- (m, 2H), 4.27-40 (m, 2H), 4.42-4.70 (m, yl]carbonyl}amino)propanoyl]- 4H), 5.00-5.08 (m, 1H), 7.17 (s, 1H), 1,4′-bipiperidin-4- 7.22-7.25 (m, 1H), 7.31-7.33 (m, 1H), yl}-4H-1,2,4-triazole-3- 7.42-7.51 (m, 2H), 7.65-7.66 (m, 1H), carboxylate 7.75 (s, 1H), 8.02 (s, 1H) (4 exchangeable protons not observed) 7 N-{(2R)-3-(7-methyl-1H- 4, 19 (400 MHz, CD₃OD) δ: ppm −0.29 to −0.19 m/z 691.6 (ES⁺) indazol-5-yl)-1-oxo-1-[4- Procedure 1 (m, 1H), 0.83-0.86 (m, 1H), 1.48- at 2.92 min, 100% (4H-1,2,4-triazol-3-yl)- 1.59 (m, 3H), 1.82-2.07 (m, 5H), 2.24- 1,4′-bipiperidin-1′- 2.35 (m, 2H), 2.46-2.60 (m, 5H), 2.63- yl]propan-2-yl}-4-(2- 2.71 (m, 1H), 2.87-3.17 (m, 7H), 3.23- oxo-1,2-dihydroquinolin- 3.41 (m, 2H), 3.59-3.63 (m, 1H), 4.08- 3-yl)piperidine-1- 4.24 (m, 2H), 4.63-4.67 (m, 1H), 7.10- carboxamide 7.12 (m, 1H), 7.22-7.25 (m, 1H), 7.32 (d, J = 8.2, 1H), 7.47-7.51 (m, 2H), 7.60-7.64 (m, 1H), 7.73 (s, 1H), 8.07 (s, 1H), 8.58- 8.61 (m, 1H) (4 exchangeable protons not observed, 2 protons obscured by solvent peak) 8 N-{(2R)-3-(7-methyl-1H- 4, 21 (400 MHz, CD₃OD) δ: ppm −0.23 to −0.18 m/z 705.6 (ES⁺) indazol-5-yl)-1-[4-(5- Procedure 1 (m, 1H), 0.83-0.87 (m, 1H), 1.52- at 3.00 min, 95% methyl-4H-1,2,4-triazol- 1.75 (m, 2H), 1.80-2.10 (m, 5H) 2.22- 3-yl)-1,4′-bipiperidin-1′- 2.38 (m, 2H), 2.41-2.60 (m, 8H), 2.65- yl]-1-oxopropan-2-yl}-4- 2.71 (m, 1H), 2.80-3.10 (m, 8H) 3.15- (2-oxo-1,2- 3.25 (m, 1H), 3.55-3.70 (m, 2H), 4.06- dihydroquinolin-3- 4.24 (m, 3H), 4.62-4.65 (m, 1H), 5.00- yl)piperidine-1- 5.03 (m, 1H), 7.21-7.23 (m, 2H), 7.31- carboxamide 7.34 (m, 1H), 7.46-7.51 (m, 2H), 7.64- 7.72 (m, 2H), 7.96-8.05 (m, 1H) (4 exchangeable protons not observed) 9 5-{1′-[(2R)-3-(7-methyl- Example 6 (400 MHz, CD₃OD) δ: ppm −0.33 to −0.27 m/z 735.7 (ES⁺) 1H-indazol-5-yl)-2-({[4- Procedure 2 (m, 1H), 0.69-0.75 (m, 1H), 1.22- at 2.33 min, 100% (2-oxo-1,2- 1.44 (m, 2H), 1.51-1.82 (m, 4H), 1.90- dihydroquinolin-3- 2.46 (m, 7H), 2.50-2.60 (m, 4H), 2.62- yl)piperidin-1- 3.17 (m, 7H), 3.50-3.70 (m, 2H), 3.94- yl]carbonyl}amino)propanoyl]- 4.24 (m, 3H), 4.52-4.65 (m, 1H), 5.06- 1,4′-bipiperidin-4- 5.09 (m, 1H), 7.15 (s, 1H), 7.21-7.26 (m, yl}-4H-1,2,4-triazole-3- 1H), 7.31-7.33 (m, 1H), 7.47-7.51 (m, carboxylic acid 2H), 7.65-7.69 (m, 1H), 7.75 (s, 1H), 7.97-8.02 (m, 1H) (5 exchangeable protons not observed)

Biological and Biophysical Methods

Cloning, Baculovirus Generation, Large-Scale Infection of Sf21 Cells and Membrane Preparation.

Human Calcitonin Receptor Like Receptor (CRLR) and human RAMP1 were cloned into Invitrogen's (ThermoFisher Scientific, UK) pFastBac dual expression vector. Transposition of CRLR/RAMP1 DNA was performed using Invitrogen's Bac-to-Bac Baculovirus Expression Systems. P0 baculovirus was generated by transfecting SF9 cells with bacmid DNA using Cellfectin® II transfection reagent (ThermoFisher Scientific, UK, catalog number 10362-100). Following P0 generation P1 virus was then generated ready for large scale infection and membrane preparation. Sf21 cells were grown in expression medium ESF921 (Expression Systems, USA, catalog number 96-001-01) supplemented with 10% heat-inactivated FBS and 1% Pen/Strep and were infected at a cell density of 2.5×10⁶ cells/mL and an MOT of 2. Expression was carried out over 48 h in a shaking incubator set at 27° C. The cell culture was centrifuged at 2,500 rcf for 10 min at 4° C. The pellets were resuspended in cold PBS supplemented with Roche's Complete EDTA-free protease inhibitor cocktail tablets (Roche Applied Sciences, catalog number 05056489001), 1 mM PMSF and 1 mM EDTA. The resuspended cell paste was then centrifuged at 3,273 rcf for 12 min at 4° C. The supernatant was discarded and the pellet frozen at −80° C. The cell pellet from a 4 L culture was resuspended in buffer containing 50 mM Hepes pH 7.5, 150 mM NaCl, 8 Roche EDTA-free protease inhibitor cocktail tablets and 1 mM PMSF. The suspension was left stirring at rt for 1 h and then homogenised for 90 s at 9,500 rpm using a VDI 25 (VWR, USA) homogeniser. The cells were then lysed using a Microfluidizer processor M-110L Pneumatic (Microfluidics, USA). After lysis, the mixture was homogenised for 90 s at 9,500 rpm and then centrifuged at 335 rcf for 10 min. The supernatant was then further ultra-centrifuged at 42,000 rpm for 90 min. After ultra-centrifugation, the supernatant was discarded and the pellet was resuspended in 50 mL (25 mL for each 2 L culture) of buffer containing 50 mM Hepes pH 7.5, 150 mM NaCl, 3 Roche EDTA-free protease inhibitor cocktail tablets and 1 mM PMSF. The suspension was then homogenised for 90 s at 9,500 rpm. The resulting membranes were then stored at −80° C.

Radioligand Binding Assay.

Human CGRP receptors (consisting of CRLR and RAMP1) expressed in insect Sf21 cell membrane homogenates were re-suspended in the binding buffer (10 mM HEPES, pH 7.4, 5 mM MgCl₂, 0.2% BSA) to a final assay concentration of 0.6 μg protein per well. Saturation isotherms were determined by the addition of various concentrations of ³H-telcagepant (Ho et al, The Lancet, 2008, 372, 2115) (in a total reaction volume of 250 μL) for 60 min at room temperature. At the end of the incubation, membranes were filtered onto a unifilter, a 96-well white microplate with bonded GF/B filter pre-incubated with 0.5% PEI, with a Tomtec cell harvester and washed 5 times with distilled water. Non-specific binding (NSB) was measured in the presence of 10 nM MK-3207 hydrochloride (CAS No. 957116-20-0). Radioactivity on the filter was counted (1 min) on a microbeta counter after addition of 50 μL of scintillation fluid. For inhibition experiments, membranes were incubated with 0.5 nM ³H-telcagepant and 10 concentrations of the inhibitory compound (0.001-10 μM). IC₅₀ values were derived from the inhibition curve and the affinity constant (K_(i)) values were calculated using the Cheng-Prussoff equation (Cheng et al, Biochem. Pharmacol. 1973, 22, 3099-3108). The pK_(i) values (where pK_(i)=−log₁₀ K_(i)) of certain compounds of the invention are tabulated below.

cAMP Functional Assay.

cAMP production following receptor activation was determined using the Homogeneous Time-Resolved Fluorescence (HTRF) cAMP dynamic-2 assay (Cisbio, France). The human neuroblastoma cell line SK-N-MC endogenously expressing the human CGRP receptor was seeded at a density of 12,500 cells/well in solid walled 96 well half area plates (Costar, Catalog Number 3688, Corning Life Sciences, Germany). After 16 h incubation at 37° C. media was removed and cells were incubated at 37° C. for 30 min in serum free media containing 500 μM IBMX (Tocris, Abingdon, UK, Catalog Number 2845) and increasing concentrations of test antagonist. Following this cells were challenged with an EC₈₀ concentration of human CGRP (0.3 nM) for a further 30 min at 37° C. and then cAMP production was determined as manufacturer's instructions before plates were read on a PheraStar fluorescence plate reader (BMG LabTech, Germany). IC₅₀ values were derived from the inhibition curve. The pIC₅₀ values (where pIC₅₀=−log₁₀ IC₅₀) were converted to a functional pK_(b) value using a modified Cheng-Prussoff equation where K_(d)=agonist EC₅₀ and L hot=agonist challenge concentration. The pK_(b) values of certain compounds of the invention are detailed in Table 3.

TABLE 3 Ex pK_(i) pK_(b) No. Name Structure average average 1 N-[(2R)-1-[4-(1H-imidazol-2-yl)- 1,4′-bipiperidin-1′-yl]-3-(7- methyl-1H-indazol-5-yl)-1- oxopropan-2-yl]-4-(2-oxo-1,2- dihydroquinolin-3-yl)piperidine-1- carboxamide

10.3 9.7 2 N-[(2R)-1-[4-(4-methyl-1H- imidazol-5-yl)-1,4′-bipiperidin-1′- yl]-3-(7-methyl-1H-indazol-5-yl)- 1-oxopropan-2-yl]-4-(2-oxo-1,2- dihydroquinolin-3-yl)piperidine-1- carboxamide

10.1 9.5 3 N-{(2R)-3-(7-methyl-1H-indazol- 5-yl)-1-oxo-1-[4-(1-propyl-1H- imidazol-2-yl)-1,4′-bipiperidin-1′- yl]propan-2-yl}-4-(2-oxo-1,2- dihydroquinolin-3-yl)piperidine-1- carboxamide

 9.8 9.4 4 N-{(2R)-3-(7-methyl-1H-indazol- 5-yl)-1-[4-(4-methyl-4H-1,2,4- triazol-3-yl)-1,4′-bipiperidin-1′- yl]-1-oxopropan-2-yl}-4-(2-oxo- 1,2-dihydroquinolin-3- yl)piperidine-1-carboxamide

10.0 9.2 5 4-(7-fluoro-2-oxo-1,2- dihydroquinolin-3-yl)-N-{(2R)-3- (7-methyl-1H-indazol-5-yl)-1-[4- (4-methyl-4H-1,2,4-triazol-3-yl)- 1,4′-bipiperidin-1′-yl]-1- oxopropan-2-yl}piperidine-1- carboxamide

 9.9 9.2 6 ethyl 5-{1′-[(2R)-3-(7-methyl-1H- indazol-5-yl)-2-){[4-(2-oxo-1,2- dihydroquinolin-3-yl)piperidin-1- yl]carbonyl}amino)propanoyl]- 1,4′-bipiperidin-4-yl}-4H-1,2,4- triazol-3-carboxylate

10.2 9.7 7 N-{(2R)-3-(7-methyl-1H-indazol- 5-yl)-1-oxo-1-[4-(4H-1,2,4- triazol-3-yl)-1,4′-bipiperidin-1′- yl]propan-2-yl}-4-(2-oxo-1,2- dihydroquinolin-3-yl)piperidine-1- carboxamide

 9.8 9.5 8 N-{(2R)-3-(7-methyl-1H-indazol- 5-yl)-1-[4-(5-methyl-4H-1,2,4- triazol-3-yl)-1,4′-bipiperidin-1′- yl]-1-oxopropan-2-yl}-4-(2-oxo- 1,2-dihydroquinolin-3- yl)piperidine-1-carboxamide

10.0 9.5 9 5-{1′-[(2R)-3-(7-methyl-1H- indazol-5-yl)-2-({[4-(2-oxo-1,2- dihydroquinolin-3-yl)piperidin-1- yl]carbamoyl}amino)propanoyl]- 1,4′-bipiperidin-4-yl}-4H-1,2,4- triazole-3-carboxylic acid

10.3 9.4

Pharmacokinetic Profiling.

The pharmacokinetic profiles of Examples and reference compounds have been assessed in male Sprague Dawley® rats via intravenous (iv), sub-cutaneous (sc) and intranasal (IN) routes of delivery, and in male Cynomolgus Monkeys via iv and sc routes of delivery. Pharmacokinetic data for Examples of the invention and a reference compound, olcegepant, are detailed in Tables 4 and 5.

Methods:

For rat studies, groups of three male Sprague Dawley® rats, typically ranging in weight between 180 and 300 g, were given a single dose of Example or reference compound via one of the following routes: iv, sc or IN, using doses, dose volumes and vehicles specified in Table 5. Prior to IN dosing rats were anaesthetised with an intramuscular dose of 25-30 mg/kg ketamine cocktail (ketamine, xylazine hydrochloride and acepromazine maleate in saline) and the dose is introduced over 20-30 s via a polyethylene PE-10 tube inserted approximately 5 mm into the nasal cavity of the rat.

For cynomolgus monkey studies, groups of three male monkeys, typically ranging in weight between 3.0 and 4.5 kg, were given a single dose of Example or reference compound via one of the following routes: iv or sc, using doses, dose volumes and vehicles specified in Table 5. Following dosing by the routes above blood samples were taken at several time points (typically pre-dose, 0.083, 0.25, 0.5 1, 2, 4, 8 and 24 h) via serial tail vein bleeds (rat) or cephalic or saphenous vein (monkey) from the animal and centrifuged to separate plasma for analysis by LC/MS/MS assay. WinNonlin v6.2 statistics software (Pharsight Corporation, California, USA) was used to generate pharmacokinetic parameters using the non-compartmental model.

TABLE 4 Rat iv pharmacokinetics Dose Dose volume Clearance (mg/kg) (mL/kg) Vehicle (mL/min/kg) olcegepant 5 1 10% DMAC + 10% 18 SolutolHS15 + 80% Saline Example 2 2 1 10% DMAC + 10% 18 SolutolHS15 + 80% Saline Example 4 2 1 10% DMAC + 10% 2 SolutolHS15 + 80% Saline Rat sc pharmacokinetics Dose Bioavail- Dose volume ability (mg/kg) (mL/kg) Vehicle (%) olcegepant 1 5 10% DMAC + 10% 48 SolutolHS15 + 80% Saline Example 2 1 2 Acidified saline 69 Example 4 2 5 10% DMAC + 10% 82 SolutolHS15 + 80% Saline Rat IN pharmacokinetics Dose Bioavail- Dose concentration, ability (mg/kg) Dose volume Vehicle (%) olcegepant 1.3 6 mg/mL, Acidified saline 8 50 μL Example 2 1 12 mg/mL, Acidified saline 25 25 μL Example 4 1 12 mg/mL, Acidified saline 27 25 μL

TABLE 5 Cynomolgus monkey iv pharmacokinetics Dose Dose volume Clearance (mg/kg) (mL/kg) Vehicle (mL/min/kg) Example 2 0.5 0.5 Acidified saline 24 Example 4 0.5 0.5 Acidified saline 15 Cynomolgus monkey sc pharmacokinetics Dose Bioavail- Dose volume ability (mg/kg) (mL/kg) Vehicle (%) Example 2 0.5 1 Acidified saline 100 Example 4 0.5 1 Acidified saline 100

Thermodynamic Solubility Profiling.

A 50 mM DMSO stock solution of test compound was prepared, and from this, a working solution of 1 mM was prepared by dilution with DMSO. The UV absorbance of working solution was scanned from 220 nm to 1000 nm to identify the wavelength maxima of test compound. The 1 mM working solution was then serially diluted in DMSO to different concentrations to determine linearity/calibration curve. To ascertain the aqueous thermodynamic solubility of test compound, samples were added to a volume of PBS buffer (pH 7.4) or Sodium Phosphate Buffer (pH 6.0) which was appropriate to generate a final concentration of 1 mg/mL if all test compound dissolved. The resulting solution was then kept on a RotoSpin shaker at 50 rpm for 24 h at rt before the solution was filtered using 0.45 micron PVDF injector filters in order to remove the insoluble fraction of the compound. Subsequently, 150 uL of the filtrate is taken for quantification using a UV spectrophotometer, acquiring the optical density of standard solutions and test compound at the same wavelength maxima. From the optical density of test compound the thermodynamic solubility is calculated using the linearity/calibration curve and expressed as micromolar (μM). Solubility profiles of certain compounds of the invention are detailed in Table 6.

TABLE 6 Reference Cpd/ Thermodynamic solubility (μM) Example pH 6 pH 7.4 olcegepant  150 431 Example 1 Not tested Not tested Example 2 2041  18 Example 3 1249  2 Example 4 1587  32 Example 5 1541 239 Example 6 Not tested Not tested Example 7  785 342 Example 8 1217 359 Example 9 Not tested Not tested 

1. A compound of formula (I):

or salts thereof, wherein R¹ is H or F and Ar¹ is an optionally substituted 5 membered heterocyclic ring containing at least two nitrogen atoms.
 2. The compound according to claim 1, wherein Ar¹ is an optionally substituted five-membered heterocyclic ring including at least two nitrogen atoms, wherein the optional substituents are selected from (C₁-C₆)alkyl, CO₂R² where R² is H or (C₁-C₃)alkyl.
 3. The compound according to claim 1, wherein R¹ is H.
 4. The compound according to claim 1, wherein Ar¹ is a five-membered heterocyclic ring including two or three nitrogen atoms, optionally substituted with (C₁-C₆)alkyl.
 5. The compound according to claim 1, wherein Ar¹ is


6. The compound according to claim 1, wherein Ar¹ is


7. The compound according to claim 1, wherein Ar¹ is


8. The compound according to claim 1, wherein the compound is selected from the group consisting of: N-[(2R)-1-[4-(1H-imidazol-2-yl)-1,4′-bipiperidin-1′-yl]-3-(7-methyl-1H-indazol-5-yl)-1-oxopropan-2-yl]-4-(2-oxo-1,2-dihydroquinolin-3-yl)piperidine-1-carboxamide; N-[(2R)-1-[4-(4-methyl-1H-imidazol-5-yl)-1,4′-bipiperidin-1′-yl]-3-(7-methyl-1H-indazol-5-yl)-1-oxopropan-2-yl]-4-(2-oxo-1,2-dihydroquinolin-3-yl)piperidine-1-carboxamide; N-{(2R)-3-(7-methyl-1H-indazol-5-yl)-1-oxo-1-[4-(1-propyl-1H-imidazol-2-yl)-1,4′-bipiperidin-1′-yl]propan-2-yl}-4-(2-oxo-1,2-dihydroquinolin-3-yl)piperidine-1-carboxamide; N-{(2R)-3-(7-methyl-1H-indazol-5-yl)-1-[4-(4-methyl-4H-1,2,4-triazol-3-yl)-1,4′-bipiperidin-1′-yl]-1-oxopropan-2-yl}-4-(2-oxo-1,2-dihydroquinolin-3-yl)piperidine-1-carboxamide; 4-(7-fluoro-2-oxo-1,2-dihydroquinolin-3-yl)-N-{(2R)-3-(7-methyl-1H-indazol-5-yl)-1-[4-(4-methyl-4H-1,2,4-triazol-3-yl)-1,4′-bipiperidin-1′-yl]-1-oxopropan-2-yl}piperidine-1-carboxamide; ethyl 5-{1′-[(2R)-3-(7-methyl-1H-indazol-5-yl)-2-({[4-(2-oxo-1,2-dihydroquinolin-3-yl)piperidin-1-yl]carbonyl}amino)propanoyl]-1,4′-bipiperidin-4-yl}-4H-1,2,4-triazole-3-carboxylate; N-{(2R)-3-(7-methyl-1H-indazol-5-yl)-1-oxo-1-[4-(4H-1,2,4-triazol-3-yl)-1,4′-bipiperidin-1′-yl]propan-2-yl}-4-(2-oxo-1,2-dihydroquinolin-3-yl)piperidine-1-carboxamide; N-{(2R)-3-(7-methyl-1H-indazol-5-yl)-1-[4-(5-methyl-4H-1,2,4-triazol-3-yl)-1,4′-bipiperidin-1′-yl]-1-oxopropan-2-yl}-4-(2-oxo-1,2-dihydroquinolin-3-yl)piperidine-1-carboxamide; and 5-{1′-[(2R)-3-(7-methyl-1H-indazol-5-yl)-2-({[4-(2-oxo-1,2-dihydroquinolin-3-yl)piperidin-1-yl]carbonyl}amino)propanoyl]-1,4′-bipiperidin-4-yl}-4H-1,2,4-triazole-3-carboxylic acid.
 9. The compound according to claim 1, wherein the compound is:


10. A method for treating a cerebrovascular or vascular disorder in a subject comprising administering to the subject a compound according to claim
 1. 11. The method of claim 10, wherein the cerebrovascular or vascular disorder is migraine without aura, chronic migraine, pure menstrual migraine, menstrually-related migraine, migraine with aura, familial hemiplegic migraine, sporadic hemiplegic migraine, basilar-type migraine, cyclical vomiting, abdominal migraine, benign paroxysmal vertigo of childhood, retinal migraine, status migrainosus, cluster headache, dialysis headache, paroxysmal hemicrania, osteoarthritis, hot flashes associated with menopause or medically induced menopause due to surgery or drug treatment, hemicrania continua, cyclic vomiting syndrome, allergic rhinitis, or rosacea, dental pain, earache, middle ear inflammation, sunburn, joint pain associated with osteoarthritis and rheumatoid arthritis, cancer pain, fibromyalgia, diabetic neuropathy, pain associated with inflammatory bowel disease—Crohn's disease, gout, complex regional pain syndrome, Behçet's disease, endometriosis pain, back pain, or cough.
 12. The method of claim 10 wherein the compound is administered via a non-oral route.
 13. The method of claim 12 wherein the non-oral route of administration is an intranasal route, a sub-cutaneous route or an intravenous route.
 14. A method of synthesizing a compound according to claim
 1. 